Probiotic Lactobacillus fermentum TSF331, Lactobacillus reuteri TSR332, and Lactobacillus plantarum TSP05 improved liver function and uric acid management-A pilot study

Metabolic-associated fatty liver disease (MAFLD) is predominantly associated with metabolic disturbances representing aberrant liver function and increased uric acid (UA) levels. Growing evidences have suggested a close relationship between metabolic disturbances and the gut microbiota. A placebo-controlled, double-blinded, randomized clinical trial was therefore conducted to explore the impacts of daily supplements with various combinations of the probiotics, Lactobacillus fermentum TSF331, Lactobacillus reuteri TSR332, and Lactobacillus plantarum TSP05 with a focus on liver function and serum UA levels. Test subjects with abnormal levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), and UA were recruited and randomly allocated into six groups. Eighty-two participants successfully completed the 60-day intervention without any dropouts or occurrence of adverse events. The serum AST, ALT, and UA levels were significantly reduced in all treatment groups (P < 0.05). The fecal microbiota analysis revealed the intervention led to an increase in the population of commensal bacteria and a decrease in pathobiont bacteria, especially Bilophila wadsworthia. The in vitro study indicated the probiotic treatments reduced lipid accumulation and inflammatory factor expressions in HepG2 cells, and also promoted UA excretion in Caco-2 cells. The supplementation of multi-strain probiotics (TSF331, TSR332, and TSP05) together can improve liver function and UA management and may have good potential in treating asymptomatic MAFLD. Trial registration. The trial was registered in the US Library of Medicine (clinicaltrials.gov) with the number NCT06183801 on December 28, 2023.


Introduction Metabolic syndrome
Metabolic syndrome refers to a cluster of risk factors that predispose individuals to cardiovascular diseases.These risk factors primarily encompass hypertension, dyslipidemia, diabetes, obesity, as well as abnormalities in uric acid levels and coagulation factors.The Third Report of the National Cholesterol Education Program Expert Panel (NCEP ATP III) in the United States defines metabolic syndrome as the presence of three or more of the following criteria in an individual.

The Relationship Between Gut Microbiota and Metabolic Syndrome
Influence of gut microbiota on metabolic disorders includes: 1) gut microbiota-mediated fermentation of dietary fiber and resistant starch leading to the production of short-chain fatty acids (SCFAs), and 2) the presence of lipopolysaccharides (LPS) on the cell membrane of Gram-negative bacteria.In situations of excessive consumption of high-fat foods, there is a substantial proliferation of Gram-negative bacteria in the intestinal tract, leading to the abundant production of lipopolysaccharides.LPS constitute the primary component of the bacterial outer membrane, serving not only to maintain the integrity of the bacterial structure but also providing a protective function.When binding with CD14/Toll-like Receptor 4 (TLR4), LPS activates inflammatory responses, leading to the secretion of various cytokines.This cascade of events results in inflammatory manifestations, increased lipogenesis, and decreased insulin sensitivity, thereby inducing the onset of metabolic disorders.Conversely, substantial intake of high-fiber foods stimulates the proliferation of beneficial bacteria involved in fiber degradation.This leads to the production of abundant short-chain fatty acids, thereby exerting enhanced physiological functions, such as immune modulation [4].

Hyperuricemia
Uric acid is the final oxidized product of purine metabolism, excreted from the body through the kidneys in the urine.Individuals with hyperuricemia may experience symptoms that can precipitate gout, characterized by severe pain in the extremity joints due to the formation of uric acid crystals.Additionally, this condition may give rise to complications such as renal disorders, urolithiasis, cardiovascular diseases, and cerebrovascular disorders.Therefore, hyperuricemia is also considered one of the risk factors for arterial sclerosis.According to the statistical analysis in 2013, there are 5.8 million patients suffering from hyperuricemia or gout in Taiwan.In general, the upper end of the normal range of serum uric acid is 360 µmol/L (6 mg/dL) for women and 400 µmol/L (6.8 mg/dL) for men.In patients with hyperuricemia and diabetes at the same time, clinical studies have shown significantly higher levels of blood pressure, blood lipids, cholesterols, BMI value, creatinine, and HbA1c than in patients with hyperuricemia only.
In other words, hyperuricemia increases the risk of getting other chronic diseases.
Hyperuricemia is a metabolic disorder without early symptoms, and most of patients are not aware of the elevation of their uric acid until full physical examinations are done by chance.

Probiotics
Lactic acid bacteria (LAB), commonly known as probiotics, are generally acknowledged as safe and widely prevalent.They are utilized or incorporated into numerous food and dietary supplement formulations.The word 'Probiotics' originates from the Greek words 'for life,' referring to beneficial living microorganisms that contribute to the well-being of the host.Probiotics, along with related dairy products, have a long history of being considered safe.The efficacy of these strains is closely tied to their strain specificity; however, not every strain exhibits identical benefits.The common beneficial effects of probiotics include alleviating lactose intolerance, preventing or attenuating Helicobacter pylori infections, mitigating intestinal disorders, and modulating immune responses.

Current state of intestinal microbiota analysis techniques
Next-Generation Sequencing (NGS), characterized by both high throughput and high precision, facilitates the accurate representation of highly complex microbial community profiles [2].The integration of metagenomics with NGS, utilizing bacterial 16S or fungal 18S rDNA, stands as the most efficient and widely employed approach for taxonomic identification and quantification of microbial communities [3].NGS is employed for the analysis of fecal samples, enabling high-throughput, extensive, and in-depth analysis.This technique offers high sensitivity in detecting and comprehending the most comprehensive and profound alterations in the human intestinal microbiota.

Objective
Due to Taiwan's geographical environment as an island, the dietary habits of the population tend to emphasize the consumption of seafood and organs.The prevalence of hyperuricemia in Taiwan is high, partly attributed to convivial cultural practices characterized by warm hospitality and frequent social drinking.Over the long term, this can lead to dysbiosis in the gut microbiota, subsequently impacting the body's metabolic functions.This clinical study primarily observes the impact of probiotic supplementation on subjects exhibiting abnormal liver function indices and elevated uric acid levels.After a supplementation period of two months, the study evaluates whether there are improvements in liver and kidney function parameters, as well as uric acid levels, through blood biochemical analysis.Additionally, the study explores other potential influences on these outcomes.

Study design
For the purpose of a trial focused on reducing uric acid levels and promoting liver health, we aim to recruit 120 participants who simultaneously exhibit elevated uric acid and elevated liver indices.Eligible participants will have baseline liver indices (GOT > 38 IU/L, GPT > 44 IU/L) and baseline uric acid values (male > 7 mg/dL, female > 6 mg/dL).The strains contained in the hepatoprotective and uric acid-lowering series (PRONULIFE® UriManage/ Quick Acid Clear®) products are Lactobacillus fermentum TSF331, Lactobacillus reuteri TSR332, and Lactobacillus plantarum TSP05, respectively.On days 0 and 60 following probiotic supplementation, subjects will undergo a comprehensive assessment, including anthropometric measurements (height, weight, body fat, visceral fat, and questionnaire-based evaluation), blood biochemical parameters (as detailed in the accompanying figure), and fecal sample collection.

Hematological biochemical parameters examination
Participants will be recruited and will sign informed consent forms upon agreement.Preprobiotic serum samples will be collected before the ingestion of probiotics.Participants will be instructed to consume the probiotics according to the trial plan for a duration of two months.Following this period, post-probiotic serum samples will be collected.A comparative biochemical analysis will be conducted, encompassing parameters such as glucose AC, GOT, GPT, BUN, Creatinine, TG, total cholesterol, HDL, and LDL.

Next-generation sequencing (NGS) for gut microbiota profiling
The fecal samples will be collected before and after probiotic consumption, following the standard operating procedure for 'Preparing 16S Ribosomal RNA Gene Amplicons for the Illumina MiSeq System.'The amplification of the V3 and V4 gene segments of 16S rRNA is conducted, and subsequent analysis is performed using Illumina MiSeq next-generation sequencing.

Bioinformatics analysis
Analysis of intestinal bacterial diversity and abundance will be derived from NGS sequences using Illumina BaseSpace with Greengenes 13.7 as the reference database.
Subsequent processing involves the utilization of CLC Genomic Workbench (Qiagen) software to generate an operational taxonomic unit (OTU) table and conduct alpha diversity and beta diversity analyses.